Structure-activity study of dihydrocinnamic acids and discovery of the potent FFA1 (GPR40) agonist TUG-469

Article abstract of DOI:10.1021/ml100106c

The free fatty acid 1 receptor (FFA1 or GPR40), which is highly expressed on pancreatic β-cells and amplifies glucose-stimulated insulin secretion, has emerged as an attractive target for the treatment of type 2 diabetes. Several FFA1 agonists containing the para-substituted dihydrocinnamic acid moiety are known. We here present a structure-activity relationship study of this compound family suggesting that the central methyleneoxy linker is preferable for the smaller compounds, whereas the central methyleneamine linker gives higher potency to the larger compounds. The study resulted in the discovery of the potent and selective full FFA1 agonist TUG-469 (29).

Full text of DOI:10.1021/ml100106c

pubs.acs.org/acsmedchemlett
Structure-Activity Study of Dihydrocinnamic Acids and
Discovery of the Potent FFA1 (GPR40) Agonist TUG-469
Elisabeth Christiansen,† Maria E. Due-Hansen,† Christian Urban,‡ Nicole Merten,§
Michael Pfleiderer, Kasper K. Karlsen,† Sanne S. Rasmussen,† Mette Steensgaard,†
Alexandra Hamacher,‡ Johannes Schmidt,§ Christel Drewke,§ Rasmus Koefoed Petersen,^
Karsten Kristiansen,^ Susanne Ullrich, Evi Kostenis,§ Matthias U. Kassack,‡ and
Trond Ulven*,†
†Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark,
‡
€
Pharmaceutical Biochemistry, Institute of Pharmaceutical and Medicinal Chemistry, University of Dusseldorf,
§
€
€
Universitatsstrasse 1, D-40225 Dusseldorf, Germany, Department of Molecular, Cellular and Pharmacobiology Institute for
Pharmaceutical Biology, University of Bonn, Nussallee 6, D-53115 Bonn, Germany, Department of Internal Medicine, Division
€
€
of Endocrinology, Diabetology, Vascular Medicine, Nephrology and Clinical Chemistry, University of Tubingen, Otfried-Muller-
^
€
Strasse 10, D-72076 Tubingen, Germany, and Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200
Copenhagen N, Denmark
ABSTRACT The free fatty acid 1 receptor (FFA1 or GPR40), which is highly expressed
on pancreatic β-cells and amplifies glucose-stimulated insulin secretion, has emerged as
an attractive target for the treatment of type 2 diabetes. Several FFA1 agonists containing
the para-substituted dihydrocinnamic acid moiety are known. We here present a
structure-activity relationship study of this compound family suggesting that the
central methyleneoxy linker is preferable for the smaller compounds, whereas the cen-
tral methyleneamine linker gives higher potency to the larger compounds. The study
resulted in the discovery of the potent and selective full FFA1 agonist TUG-469 (29).
KEYWORDS Diabetes, FFA1, FFAR1, GPR40, fatty acids, drug discovery
ith an estimated 285 million cases worldwide and
an expected 480 million cases by 2030, diabetes
represents a threat to the economy and human
administration, in contrast to fatty acids that resulted in impaired
insulin secretion.15 This outcome is also supported by several
other recent studies.16-18 Thus, although the issue is still con-
troversial, most evidence currently supports FFA1 agonists as
promising for the treatment of T2D.
Both saturated and unsaturated FFAs with more than
10 carbon atoms act as agonists on FFA1.4-6,8,9 The ability
of FFA1 to stimulate insulin secretion at elevated glucose
levels attracted immediate attention, and several series of
FFA1 agonists as well as some antagonists have since been
described, many of these containing the para-substituted
dihydrocinnamic acid or the corresponding thiazolidinedione
moiety (Figure 1).15,19-27 Herein, we describe structure-
activity investigations around the lead compound 1 and the
para-substituted dihydrocinnamic acids, which resulted in the
identification of the potent agonist 29 (TUG-469).
It was discovered in 2003 that several thiazolidinedione
PPARγ agonists, including the antidiabetic drug rosiglitazone
(Figure 1), also possess FFA1 agonistic activity.6 Presuming
that thiazolidinediones and FFAs share a common binding
site within FFA1, which has now been confirmed,28 we
included structural information from the thiazolidinedione
agonists in the design of a small library of carboxylic acids to
W
well being, and the development of improved therapies is
urgent.1,2 Recently, the receptor GPR40, now renamed to
FFA1,3 was found to be activated by medium- and long-chain
fatty acids, to be highly expressed on pancreatic β-cells, and to
potentiate glucose-stimulated insulin secretion (GSIS).4-6 FFA1
is also present in the endocrine cells of the gastrointestinal tract
and has been reported to mediate free fatty acid (FFA) stimu-
lated secretion of GLP-1 and GIP.7 Thus, the receptor appears to
act as an important nutrient sensor and metabolic regulator and
to stimulate insulin secretion by direct action on the β-cells and
possibly also indirectly via the incretin system. FFA1 is of
considerable interest as a new target for the treatment of type
2 diabetes(T2D) since insulin secretion is promoted without the
associated risk of hypoglycemia.8,9
FFAs have long been known to potentiate GSIS, but they are
also linked to β-cell dysfunction,10 and it is unclear at present
whether FFA1 mediates one or both of these effects. Whereas
some studies with FFA1-deficient mice indicate that both effects
were mediated by the receptor and that FFA1 antagonists may
therefore be of therapeutic interest for T2D,11 , 1 2 other investiga-
tions confirmed the potentiation of GSIS but found no evidence
for lipotoxic effects.13,14 Tan and co-workers demonstrated
that a selective FFA1 agonist (compd B, Figure 1) continued to
promote GSIS with no indications of lipotoxicity after chronic
Received Date: May 10, 2010
Accepted Date: June 21, 2010
Published on Web Date: July 02, 2010
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Scheme 1a
a Reagents and conditions: (a) MeOH, HCl (cat.). (b) Benzyl bromide, K2CO3, acetone. (c) LiOH, THF, H2O. (d) ArB(OH)2, Pd(OAc)2, K2CO3, PEG-400,
45 ꢀC, 2-24 h. (e) Benzaldehyde, NaBH(OAc)3, CH2Cl2.
Scheme 2a
a Reagents and conditions: (a) Ag2SO4, I2, CH2Cl2. (b) Phenylacetylene,
Pd(PPh3)4, CuI, DIPEA, DMF, 65 ꢀC. (c) NaOMe, THF, reflux.
Figure 1. Thiazolidinedione and dihydrocinnamic acid FFA1
agonists.6,15,19,22,23
.
Table 1. Exploration of the Central Linker and Conformational
Constraints
mimic constrained fatty acids in the search for more potent
and selective FFA1 agonists. The library, consisting of com-
mercial and readily synthesized compounds, resulted in
several active compounds including the two outstanding hits
TUG-14, which was optimized to TUG-424 (Figure 1),23 and
TUG-20 (1), which served as the lead structure for the work
presented here. The disclosure of GW9508 and related
compounds by GSK directed our attention to the central
linker unit, and we set out to investigate if the methyleneoxy
or the methyleneamino linker was preferred. The initial hit 1
and analogues were synthesized as outlined in Scheme 1.
Biaryl analogues were prepared by Suzuki coupling of the
corresponding halobenzyloxy ester or the free halobenzy-
lamino acid using a practical protocol with Pd(OAc)2 and
K2CO3 in PEG-400 in open air. Synthesis of the constrained
benzofurane 8 is outlined in Scheme 2.
Replacing the central oxygen of 1 by a nitrogen (2) pro-
duced a compound with somewhat reduced activity in the
calcium mobilization assay (Table 1). We were interested to
probe if the -CH2X- linker functioned only as a flexible
spacer between the central and the terminal benzene rings;
thus, compounds 3 and 4 with inverted linkers were synthe-
sized and evaluated. Both compounds turned out more
than an order of magnitude less potent than the correspond-
ing 1 and 2, indicating a key role of the linker. Replacing
the central heteroatom by a methylene group (5) produces a
pure hydrocarbon tail more closely resembling the endogenous
hFFA1 (Ca2þ
pEC50 (efficacy)a LE -Δgb at 100 μMc
)
PPARγ %
compd
Y
X
R
1
2d
CH2
O
H
H
H
H
H
H
I
6.34 ( 0.02 (100)
6.12 ( 0.03 (89)
5.20 ( 0.03 (88)
4.67 ( 0.01 (101)
5.44 ( 0.04 (116)
5.48 ( 0.02 (106)
4.90 ( 0.02 (81)
5.61 ( 0.07 (92)
6.70 ( 0.02 (108)
0.46
0.42
0.38
0.34
0.39
0.38
0.34
0.38
0.48
7
CH2
O
NH
CH2
CH2
CH2
O
7
3
NA
NA
4
NH
5
CH2
CH(Me)
CH2
6
7
O
42
8
9e
NA
CtC
H
a Efficacy is given as % response relative to 10 μM 1. b LEs were
calculated from the formula -Δg = RT ln(EC50), presuming that EC50
approximates the dissociation constant. Values are given in kcal mol-1
per nonhydrogen atom.29 c Activation relative to 1 μM rosiglitazone.
d Previously reported (pEC50 = 5.1 ( 0.4).21 e Previously reported.23
FFA ligands. Despite increased lipophilicity, the compound
turned out to be significantly less potent than 1 and 2, indicat-
ing that the methyleneoxy and the methyleneamino linkers
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play specific roles in the recognition of the receptor binding
site by the ligands. A methyl substituent on the methylene-
oxy linker (6) resulted in significant loss of activity, as did
an iodo substituent on the central ring vicinal to the
benzyloxy (7).
Table 2. Substituted 4-Benzyloxy and 4-Benzylamino Dihydro-
cinnamic Acids
The specific effect of the central linkers might be due
to hydrogen bond interactions, conformational effects, or
overall effects on the dipolar momentum. Conformational
analysis revealed that the methyleneoxy (1) and in particular
the methyleneamino (2) are preferentially oriented in the
plane of the central benzene ring, whereas the inverted
linkers (3 and 4) and the ethylene linker (5) are preferentially
oriented orthogonally to the central benzene. The benzo-
furane derivative 8 (Scheme 2), designed as a constrained
planar analogue of 1, turned out almost an order of magni-
tude less active but still demonstrates that the receptor
binding pocket can accommodate the conformation with
the phenyl group extended in the plane of the central ring.
Notably, replacing the flexible methyleneoxy or methyl-
eneamino with an alkyne (9) results in a significant increase
in potency up to pEC50 = 6.70 and confirms that the binding
pocket can accommodate the fully extended molecule.23
An initial screen of the terminal ring with bromo substitu-
tents (10-13) demonstrated that substitution is tolerated in
all positions and preferred in the meta-position, whereas the
polar 3-nitrile analogue (14) indicated that the meta-substituent
should be nonpolar to increase potency (Table 2). The nonpolar
3-CF3 indeed produced a significant improvement in analogues
15 and 16.
hFFA1 (Ca2þ
pEC50 (efficacy)a LE -Δgb at 100 μMc
)
PPARγ %
compd
R2
X
N
GW9508d 3-OPh
compd Be
7.54 ( 0.04 (103)
6.75 ( 0.04 (103)
6.26 ( 0.03 (98)
6.35 ( 0.04 (99)
7.05 ( 0.02 (99)
6.76 ( 0.04 (110)
6.28 ( 0.02 (102)
6.73 ( 0.05 (102)
7.08 ( 0.03 (103)
7.24 ( 0.03 (98)
6.69 ( 0.03 (98)
6.74 ( 0.03 (100)
6.04 ( 0.05 (97)
6.38 ( 0.02 (92)
6.87 ( 0.05 (105)
7.27 ( 0.02 (101)
5.72 ( 0.04 (97)
5.97 ( 0.02 (97)
6.31 ( 0.03 (86)
6.90 ( 0.04 (92)
7.30 ( 0.05 (112)
7.73 ( 0.04 (114)
6.92 ( 0.04 (105)
7.16 ( 0.03 (109)
0.40
0.34
0.43
0.43
0.49
0.46
0.41
0.40
0.42
0.38
0.35
0.35
0.33
0.34
0.38
0.40
0.31
0.33
0.33
0.36
0.39
0.41
0.35
0.36
54
10
11
4-Br
O
O
O
N
O
N
O
O
O
N
O
N
O
N
O
N
O
N
O
N
O
N
2-Br
12
13
14
15
16
17
18
19f
20g
21h
22
23
24
25
26
27
28
29
30
31
3-Br
19
27
3-Br
3-CN
3-CF3
3-CF3
3-OPh
4-OPh
4-OPh
4-Ph
4-Ph
3-Ph
3-Ph
2-Ph
2-Ph
51
23
34
NA
23
25
33
--i
15
31
37
86
37
51
GW9508 is central in the series and was included to enable
direct comparison under the same conditions.19-21 We also
synthesized the potent thiazolidinedione FFA1 agonist disclosed
by Merck (compd B) to directly compare our compounds to
one with established efficacy in animal models.15,25
3-(4-MeC6H4)
3-(4-MeC6H4)
3-(2-MeC6H4)
3-(2-MeC6H4)
3-(2-MeOC6H4)
3-(2-MeOC6H4)
In contrast to compounds with smaller benzyl substituents
for which the methyleneoxy analogues were more potent (1/2,
12/13, and 16/15), the methyleneoxy analogue of GW9508
(17), carrying a larger meta-phenoxy substituent, turned out
less potent than GW9508. As expected, the corresponding
para-phenoxy analogues 18 and 19 exhibited lower potency.
All N/O pairs of the ortho-, meta-, and para-biphenyl (20-25)
compounds were synthesized and tested, confirming the
meta-position as the generally preferred and the methylene-
amino linker as preferred for the larger aromatic substituents,
which is advantageous as the methyleneamino linker pro-
duces less lipophilic compounds, on average with calculated
log P 0.4 units lower than the corresponding methyleneoxy
compounds. A 2-methyl substituent on the meta-phenyl (28
and 29) resulted in significant further increase of activity for
both linkers, whereas the corresponding 4-methyl substituent
(26 and 27) rather decreased activity. The 2-methoxy com-
pounds 30 and 31 exhibit activities very close to the unsub-
stituted 22 and 23, suggesting that the increased activity of 28
and 29 might be a consequence of increased lipophilicity and
a close fit around the 2-methyl in the binding site.
a Efficacy is given as % response relative to 10 μM 1. The maximal
response of 1 was determined to 123% relative to linoleic acid. b LEs
were calculated from the formula -Δg = RT ln(EC50), presuming that
EC50 approximates the dissociation constant. Values are given in kcal
mol-1 per nonhydrogen atom.29 c Activation relative to 1 μM rosiglita-
zone. d Reported previously (pEC50 = 7.32 ( 0.03, 7.19 ( 0.54, and
7.1 ( 0.2).19-21 e Reported previously (EC50 = 88 and 10 nM).15,25
f Reported previously (pEC50 = 6.71).21 g Tested with 0.05% BSA because
of poor solubility. h Reported previously (pEC50 = 6.6 ( 0.2).21 i Toxic at
100 μM and 15% activation at 10 μM.
from 0.46 for 1 to 0.41 for 29 (Tables1 and 2), maintenanceof
a value >0.4 is satisfactory.
The lead compound 1, GW9508, and the most potent
methyleneoxy analogue 28 and methyleneamine analogue
29 were tested in a dynamic mass redistribution (DMR)
assay to obtain integrated cellular response profiles of FFA1
transfected cells.30 Dose-response curves from DMR assays
(Figure 2) give pEC50 values that correlate well with the
calcium data (Table 2).
None of the compounds have significant activity on the
related receptors FFA2 (GPR43) or FFA3 (GPR41). Because
the antidiabetic target PPARγ, like FFA1, is activated by fatty
Ligand efficiency (LE) reflects the contribution of each
nonhydrogen atom to the activity of a compound and has
become a widely accepted measure to avoid oversized mole-
cules from the optimization process.29 Although the LE dropped
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Figure 3. Effect of 29 on insulin secretion from the pancreatic rat
INS-1E β-cells at basal and stimulatory glucose concentrations.
Shown are mean values ( SEMs (n = 3). The asterisk indicates
significance to 12 mM glucose (p < 0.001).
selectivity over relevant receptors, and the confirmed ability
to increase GSIS in a pancreatic β-cell line indicate that 29
has potential for further development and will be a useful
tool in future studies of FFA1.
SUPPORTING INFORMATION AVAILABLE Synthetic pro-
cedures and compound characterization, procedures for calcium
assay, DMR assay, PPARγ assay, and insulin secretion assays.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 2. DMR analysis of FFA1 agonists. (a) hFFA1 transfected
1321N1 cells were challenged with the indicated concentrations of
29 (n > 2), and the wavelength shift (pm) was monitored over time
as a measure of receptor activation. (b) Dose-response curves:
GW9508 pEC50 = 7.06 ( 0.11 (red line); 1 pEC50 = 6.08 ( 0.15
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Tel: þ45 6550 2568. Fax: þ45 6615 8780. E-mail:
ulven@ifk.sdu.dk.
(black line); 28 pEC50 = 6.58 ( 0.09 (gold line); and 29 pEC50
7.46 ( 0.06 (blue line).
=
acids and thiazolidinediones, it is reasonable to expect that
FFA1 ligands may also possess PPARγ activity. Most of the
compounds turned out to exhibit PPARγ agonist activity at
100 μM (Tables 1 and 2), but none show significant activity at
1 μM, and all compounds are >100-fold selective for FFA1.
Being carboxylic acids and related to FFAs, it is natural to
expect that the compounds might bind strongly to serum
albumin. The activities of 29 and 28 as FFA1 agonists were,
however, only moderately affected by increasing amounts of
BSA, with pEC50 values of 7.83 ( 0.04 and 7.09 ( 0.04,
respectively, in the presence of 0.05% BSA and 7.35 ( 0.03
and 6.25 ( 0.03 with 0.5% BSA. The effect of 29 on insulin
secretion was examined in the rat β-cell line INS-1E. Glucose
(12 mM) stimulated insulin secretion 2.7-fold, and 29 was
competent to amplify GSIS by 55% but did not affect insulin
secretion at low glucose concentrations (Figure 3).
In conclusion, structure-activity relationships of para-
substituted dihydrocinnamic acid FFA1 agonists have been
explored with focus on the central linker and terminal
aromatic ring. The results indicate that the central methyle-
neoxy linker is preferred for compounds with small substit-
uents on the terminal phenyl, whereas the methyleneamino
linker appears to be preferred for compounds with larger
aromatic substituents. The effort converted the initial lead
compound 1 into the potent and selective agonist 29, a
compound closely related to GW9508, while maintaining
LE > 0.4 kcal/mol per nonhydrogen atom. A high activity in
the label-free DMR assay, satisfactory tolerance to BSA, high
Funding Sources: We thank the Danish Council for Independent
Research/Technology and Production (Grant 09-070364) and the
German Research Foundation (DFG Grants UL140/7-1 and GRK1302/1)
and the Dr. Hilmer Foundation for financial support.
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2010 American Chemical Society
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DOI: 10.1021/ml100106c ACS Med. Chem. Lett. 2010, 1, 345–349
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